Preclinical safety and efficacy of andexanet alfa in animal models.

Essentials There is currently no approved reversal agent for factor Xa (FXa) inhibitors Andexanet alfa has been developed to reverse the anticoagulant effects of FXa inhibitors Andexanet reduced blood loss and anticoagulation markers in rivaroxaban-anticoagulated rabbits Andexanet was well tolerated in monkeys and rats, with no evidence of prothrombotic activity SUMMARY: Background Andexanet alfa is a recombinant modified form of factor Xa (FXa), designed to bind to and reverse the anticoagulant activity of FXa inhibitors. Objectives To evaluate the ability of andexanet to reverse the anticoagulant activity of rivaroxaban, and assess its pharmacokinetics (PK) and toxicity in animal models. Methods The effects of andexanet on blood loss, anti-FXa activity, rivaroxaban unbound plasma concentrations and other coagulation parameters were assessed in a rabbit liver laceration 'treatment' model. Andexanet was administered 10 min after blood loss was initiated. The toxicity of repeated administration of andexanet (up to 60 mg kg-1 day-1 ) was assessed in cynomolgus monkeys. PK parameters were evaluated in rats and monkeys. Results Excess blood loss due to anticoagulation with rivaroxaban was significantly decreased by a single intravenous bolus administration of andexanet at 35 and 75 mg per rabbit, by 75% and 63%, respectively. This correlated with dose-dependent decreases in the unbound fraction of rivaroxaban and anti-FXa activity. Co-administration of rivaroxaban had no significant impact on the PK parameters of andexanet. Andexanet (up to 60 mg kg-1 day-1 ) was well tolerated in monkeys, with no accumulation of andexanet or rivaroxaban. There was a single occurrence of anaphylaxis, which resolved after treatment with diphenhydramine and epinephrine. There was no histological evidence of prothrombotic activity with high-dose andexanet compared with vehicle control, as measured by clot and fibrin deposition in all major organs. Conclusions These data suggest that andexanet is a promising therapy for the reversal of FXa inhibitor-induced anticoagulation, supporting clinical studies in humans.

Allometric pharmacokinetic scaling: towards the prediction of human oral pharmacokinetics.

PURPOSE: To evaluate (1) allometric scaling of systemic clearance (CL) using unbound drug concentration, (2) the potential usage of brain weight (BRW) correction in allometric scaling of both CL and oral clearance (CL/F). METHODS: Human clearance was predicted allometrically (CLu = a x W(biv)) using unbound plasma concentration for eight Parke-Davis compounds and 29 drugs from literature sources. When the exponent b(iv) was higher than 0.85, BRW was incorporated into the allometric relationship (CLu*BRW = a x W(biv)). This approach was also applied to the prediction of CLu/F for 10 Parke-Davis compounds. Human oral t1/2, Cmax, AUC, and bioavailability were estimated based on allometrically predicted pharmacokinetic (PK) parameters. RESULTS: Human CL and CL/F were more accurately estimated using unbound drug concentration and the prediction was further improved when BRW was incorporated into the allometric relationship. For Parke-Davis compounds, the predicted human CL and CL/F were within 50-200% and 50-220% of the actual values, respectively. The estimated human oral t1/2, Cmax, and AUC were within 82-220%, 56-240%, and 73-190% of the actual values for all 7 compounds, suggesting that human oral PK parameters of those drugs could be reasonably predicted from animal data. CONCLUSIONS: Results from the retrospective analysis indicate that allometric scaling of free concentration could be applied to orally administered drugs to gain knowledge of drug disposition in man, and to help decision-making at early stages of drug development.

Enantiomeric determination of amphetamine and methamphetamine in urine by precolumn derivatization with Marfey's reagent and HPLC.

An analytical method was developed for enantiomeric determination of amphetamine and methamphetamine in human urine. The enantiomers were isolated from urine by solid-phase extraction, and diastereomers were formed by derivatization with the chiral Marfey's reagent (1-fluoro-2,4-dinitrophenyl-5-l-aniline amide). The diastereomers were separated by reversed-phase high-performance liquid chromatography in a water/methanol mobile phase and detected by absorbance spectrophotometry at 340 nm. Linear standard curves were obtained for all four enantiomers over a concentration range of 0.16-1.00 mg/L in urine. The detection limit was 0.16 mg/L urine for each enantiomer, and the limit of quantitation was 0.40 mg/L. The urine of 10 decedents was analyzed by this method and by a previously published precolumn derivatization procedure using (-)-1-(9-fluorenyl)ethyl chloroformate (FLEC) as the derivatizing agent and fluorescence detection. Comparison of the results of the two methods by linear regression showed comparable results for both d-amphetamine and d-methamphetamine. Neither method detected the presence of the l-enantiomers in the urine samples.

p-Hydroxymethamphetamine enantiomer pharmacokinetics and metabolism in rats: absence of N-demethylation.

p-hydroxymethamphetamine (OHMAP) is one of the major metabolites of the widely abused drug methamphetamine (MAP). The demethylation of OHMAP to p-hydroxyamphetamine (OHAP) has been shown in vitro but has never been reported in vivo. The disposition kinetics as well as the metabolism of OHMAP was investigated employing a sensitive HPLC method which can separate the enantiomers of OHMAP and OHAP. Both conjugated and unconjugated forms of these compounds can be quantitated. Male Sprague-Dawley rats were given an iv bolus of racemic OHMAP (20 mg kg-1) and serum and urine samples were collected at selected times. The serum concentration-time data for OHMAP enantiomers could be described by a biexponential equation. The clearance of D-OHMAP (93.5 mL min-1 kg-1) was slightly, but statistically significantly, greater than that of the L-enantiomer (83.9 mL min-1 kg-1). The steady-state volumes of distribution of L- and D-OHMAP were (mean +/- SD) 3.15 +/- 0.84 and 4.23 +/- 1.76 L kg-1, respectively. No significant concentrations or amounts of OHAP enantiomers could be detected in any serum or urine sample. Rats excreted more unchanged L-OHMAP (34%) than D-OHMAP (29%). In contrast, more conjugated D-OHMAP (57%) was recovered compared to the conjugated L-OHMAP (52%). The results suggest that there is slight stereoselectivity in the disposition of OHMAP enantiomers. The N-demethylation product (OHAP) was not produced in vivo.

Comparative pharmacokinetics and interspecies scaling of amphotericin B in several mammalian species.

This study employed several interspecies scaling methods, to evaluate the applicability of extrapolating to man, pharmacokinetic information obtained from animals for amphotericin B, an anti-fungal drug. Pharmacokinetic parameters from four animal species (mouse, rat, monkey and dog) and man were obtained from the literature or from analysis of data reported in the literature. The allometric relationships (obtained from four animal species) as a function of species body weight (W; kg) for systemic clearance per maximum life span potential (CLS/MLP), steady-state volume of distribution (VSS), apparent volume of distribution (V beta) and volume of the central compartment (VC) were: 5691W1.096; 2.46W0.839; 3.08W0.948 and 1.07W0.965, respectively. The allometric relationships for half-life (h) and mean residence time (h) did not scale well with body weight. The prediction of pharmacokinetic parameters in man from the allometric equations do not always agree with those reported in the literature which are based upon a limited number of studies with few human subjects. The plasma concentration-time profiles from these animals were adjusted by normalizing the concentration with dose/W0.948, and re-plotted on different pharmacokinetic time scales. The syndesichrons plot produced an almost superimposable profile of adjusted concentrations as a function of adjusted time among the four species.

Simple apparatus for serial blood sampling in rodents permitting simultaneous measurement of locomotor activity as illustrated with cocaine.

A simple device has been developed for serial venous blood sampling which permits the simultaneous measurement of locomotor activity in the freely moving rat. The device can be easily constructed from routine laboratory material and it does not interfere with the light beams used to measure locomotor activity. The device, in conjunction with an activity cage, has been applied to the combined pharmacokinetic and pharmacodynamic modeling of cocaine. The relationship between the locomotor activity following a single short iv infusion of cocaine (5 mg/kg) and cocaine plasma concentrations can be adequately described by the Sigmoid-E(max) model. Further, the relationship between activity and time can be described by the same model coupled with an effect compartment. These results suggest the applicability of the device in facilitating pharmacokinetic/pharmacodynamic modeling of drugs that affect locomotor activity.

Influence of activated charcoal on the disposition kinetics of methamphetamine enantiomers in the rat following intravenous dosing.

Methamphetamine (MAP) is a central nervous system stimulant that is widely abused by populations of several countries. There is no specific antidote for the treatment of an overdose. Activated charcoal administered orally has been used to enhance the systemic elimination of certain toxic substances via "gastrointestinal dialysis". The results of in vitro studies have shown that MAP can be rapidly adsorbed from solution by activated charcoal. We have evaluated the effect of a single oral dose of activated charcoal on the disposition kinetics of MAP following a short iv infusion. Male Sprague-Dawley rats were given an oral dose of activated charcoal (Actidose-aqua, 1 g/kg) 10 min before a short iv infusion of racemic MAP; whereas the control group was given an equivalent volume of water. Enantiomers of MAP and metabolites in serum and urine were analyzed by an enantiomer-specific method which employed HPLC and detection of a fluorescent derivative. There were no differences in any of the disposition parameters between the two groups. Within each group, the clearance (CLs) of l-MAP was greater than that of d-MAP. However, there were no differences in the steady-state volume of distribution (Vss). The CLs (mL/(min kg)) and Vss (L/kg) values for l- and d-MAP in the control group were (mean +/- SD): 55.8 +/- 20.4, 48.7 +/- 17.9, 2.64 +/- 1.16, and 2.90 +/- 1.36, respectively. The corresponding values in the charcoal-pretreated group were (mean +/- SD): 57.4 +/- 23.4, 51.1 +/- 20.7, 2.79 +/- 1.32, and 2.98 +/- 1.47. These results suggest that oral activated charcoal does not enhance the elimination of MAP from the body.

Separation and quantitation of the enantiomers of methamphetamine and its metabolites in urine by HPLC: precolumn derivatization and fluorescence detection.

To study the disposition kinetics of methamphetamine (MAP), we have developed a sensitive high-performance liquid chromatographic (HPLC) assay to quantitate the enantiomers of MAP and its major metabolites, amphetamine (AP), p-hydroxymethamphetamine (p-OH-MAP), and p-hydroxyamphetamine (p-OH-AP), the latter two of which are hydroxylated metabolites, in rat urine. To determine conjugated hydroxylated metabolites, urine samples were treated with beta-glucuronidase. Both hydrolyzed and nonhydrolyzed p-OH-MAP and p-OH-AP were extracted into ethyl acetate and back extracted with 0.05M HCl. To determine MAP and AP, urine samples were extracted with benzene, followed by back extraction into 0.05M HCl. The acid layer was collected, and to it was added (-)-1-(9-fluorenyl)ethyl chloroformate (FLEC) for the derivatization of MAP and its metabolites. Derivatization was allowed to proceed for 24 h at room temperature. The derivatized products were separated on a C18 column with a mobile phase consisting of acetate buffer (pH 3.6)-acetonitrile-tetrahydrofuran. Quantitation was achieved using a fluorescence detector at an excitation wavelength of 265 nm and an emission wavelength of 330 nm. Linear standard curves were obtained over the concentration range of 5-100 ng/mL. The interday and intraday coefficients of variation for the assay for all eight enantiomers at 10 and 75 ng/mL were less than 13%. The detection limit was 5 ng/mL or 0.5 ng on-column.(ABSTRACT TRUNCATED AT 250 WORDS)

Sensitive enantiomer-specific high-performance liquid chromatographic analysis of methamphetamine and amphetamine from serum using precolumn fluorescent derivatization.

In order to study the stereoselective disposition of methamphetamine (MAP), a widely abused drug, we have developed a sensitive HPLC assay to separate and quantitate the enantiomers of MAP and amphetamine (AP) in rat serum. Serum samples to which was added aniline sulfate (internal standard) were alkalized with 0.02 M carbonate buffer (pH 10.6) and extracted with ethyl acetate. Following back extraction with hydrochloric acid, neutralization, and reconstitution, the sample was derivatized with (-)-fluorenylethyl chloroformate overnight at room temperature. The derivatized products were separated following injection onto a reversed-phase C18 column. The mobile phase consisted of 0.02 M acetate buffer-acetonitrile-tetrahydrofuran (46:39:15, v/v). The fluorescent intensity of the effluent was monitored at excitation and emission wavelengths of 265 and 330 nm, respectively. The derivatized aniline, R-, S-AP, R- and S-MAP had retention times of 21.0, 22.6, 23.6, 27.7 and 29.0 min, respectively. Linear standard curves were obtained over the concentration range of 5-250 ng/ml. The inter-day and intra-day coefficients of variation for the assay of all four compounds at 12.5, 50.0 and 250 ng/ml were in the range of 2.1-18.6%. The method was applied to quantitate the concentrations of MAP and AP enantiomers in rat serum following a short term intravenous infusion of racemic MAP (15 mg/kg). There were no differences in serum concentrations of MAP enantiomers but the concentrations of S-AP were consistently greater than those of R-AP. These data suggest a stereoselective disposition for the formation and/or elimination of amphetamine.

Disposition kinetics of d- and l-amphetamine following intravenous administration of racemic amphetamine to rats.

Amphetamine (AP), a chiral drug, displays stereoselective differences in biological action. The effect of stereochemistry on the disposition kinetics of the enantiomers has not been thoroughly studied. We examined the disposition kinetics of AP in rats using a sensitive precolumn derivatization HPLC method that can separate the enantiomers of AP and its metabolites. Male Sprague-Dawley rats were given a short intravenous infusion of racemic AP (15 mg/kg). The systemic and renal clearances, steady-state volume of distribution, and terminal half-life for l-AP were (mean +/- SD), respectively: 65.6 +/- 9.25 ml/min.kg; 15.4 +/- 2.55 ml/min.kg; 4.33 +/- 0.71 liters/kg; and 0.96 +/- 0.13 hr. The corresponding values for d-AP were: 50.8 +/- 6.88 ml/min.kg; 12.5 +/- 2.02 ml/min.kg; 3.84 +/- 0.55 liters/kg; and 1.12 +/- 0.09 hr. There are statistically significant differences between the enantiomers in all pharmacokinetic parameters except half-life. About 40% of the l-AP dose was excreted in urine as l-p-hydroxyamphetamine and 24% as intact drug. The corresponding values for d-AP were 32% and 26%, respectively. p-Hydroxyamphetamine was primarily excreted into urine as the conjugated form. These data indicate stereoselective differences in the pharmacokinetics of the enantiomers of AP after administration of racemic drug in the rat.

Inhibitory effect of 4-methylpyrazole on antipyrine clearance in rats.

Pyrazole and 4-methylpyrazole (4-MP) are potent, effective inhibitors of alcohol dehydrogenase. Pyrazole and its derivatives also have been shown to affect the cytochrome P-450 dependent monooxygenase system. This study was performed to investigate the effect of 4-MP on the disposition kinetics of antipyrine (AP). Groups of male Fisher 344 rats were given an ip injection of 4-MP (100 mg/kg) or 4-MP HCl (equivalent to 4-MP 100 mg/kg) or an equivalent volume of saline. AP (20 mg/kg) was injected intravenously via the jugular vein catheter 30 minutes later. Blood samples were collected upto 24 hours and assayed by HPLC. 4-MP pretreatment significantly decreased AP clearance from 0.490 +/- 0.032 to 0.095 +/- 0.014 (4-MP HCl) and 0.076 +/- 0.008 (4-MP) L/hr.kg (p less than 0.01). The volume of distribution of AP decreased from 0.82 +/- 0.07 to 0.65 +/- 0.06 (4-MP HCl) and 0.56 +/- 0.04 (4-MP) L/kg (p less than 0.05). Mean residence time increased from 1.68 +/- 0.09 to 6.91 +/- 0.58 (4-MP HCl) and 7.39 +/- 0.56 (4-MP) hr (p less than 0.01). These results demonstrate a significant inhibitory effect of 4-MP on the cytochrome P-450 isozyme(s) which is responsible for AP metabolism in intact animals.